Recovery In Broth Research Articles

Roles of cell cycle phases and DNA end structures in determining requirements for the Yku, Mrx and Ligase IV complexes in nonhomologous end‐joining repair of plasmid DNA

Deoxyribonucleic acid (DNA) can be damaged through a variety of different damaging agents such as free radicals, radiation and environmental chemicals. Types of damage include single‐strand lesions such as base or sugar modifications and more deleterious lesions such as interstrand crosslinks and DNA double‐stranded breaks (DSBs). Unrepaired or misrepaired DSBs in a cell can lead to mutations and chromosome rearrangements, potentially leading to loss of cell function and even cell death. There are two independent pathways used to repair DSBs in DNA; these pathways are homologous recombination (HR) and nonhomologous end‐joining (NHEJ). NHEJ involves the functions of three major protein complexes. In the budding yeast Saccharomyces cerevisiae, these assemblages include the Yku, Mrx and DNA ligase IV complexes. Each complex has a specific function. Yku (Yku70 and Yku80) protects the broken ends from degradation and recruits other repair proteins. Mrx, composed of Mre11, Rad50 and Xrs2, may act to tether the ends together and perform nuclease processing of the ends if necessary. The ligase IV complex, containing Dnl4 and Lif1 and possibly Nej1, performs the final step, ligating the two broken ends of DNA thus ending the NHEJ process.Repair of DSBs by NHEJ is frequently analyzed using plasmid‐based assays. In these experiments, a plasmid with a single DSB is transformed into cells and the efficiency of repair/recircularization inside cells is monitored. The region around the DSB site is designed so that it lacks homology with the host cell's chromosomal DNA to prevent repair by HR. An uncut plasmid is usually also transformed into the cells to normalize repair efficiencies to simple cell transformation efficiencies. Many past studies involving NHEJ repair using plasmid assays have observed that repair in yku, mrx, and dnl4 mutants is reduced similarly, typically 10–30 fold, suggesting equal importance of the three major complexes in the pathway. Using a high efficiency yeast transformation method involving treatment with dithiothreitol (DTT) and inclusion of a rich broth recovery step, we observed that repair of broken plasmids with 5′ single‐stranded DNA overhangs (4 nt long) was strongly reduced in yku mutants (yku70 or yku80) and DNA ligase IV mutants (dnl4, lif1, or nej1), but not in mrx strains (mre11, rad50 or xrs2). The accuracy of repair was also strikingly different among mrx cells vs. yku and dnl4 mutants. By contrast, efficiency and accuracy of repair of plasmids containing 3′ overhangs was reduced to similar levels in yku70, mre11 and dnl4 mutants. Since NHEJ repair efficiency is most active in G1 phase, we have also assessed the differential repair of broken plasmids in yku, mrx and dnl4 mutants in cultures with different proportions of G1 cells. These experiments have included mid‐log phase, early stationary phase, stationary phase and alpha factor‐arrested G1 phase cells. Work utilizing linear plasmids containing different end sequences and structures will be described.Support or Funding InformationNational institute of Health (NIH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Densely adherent growth mode, rather than extracellular polymer substance matrix build-up ability, contributes to high resistance of Staphylococcus epidermidis biofilms to antibiotics

(i) To evaluate the role of the adherent growth mode and extracellular polymer substance build-up in biofilm resistance to antibiotics. (ii) To re-assess various mechanisms leading to biofilm resistance to antibiotics. We compared the biofilm MICs, biofilm MBCs using the viable count method, biofilm MBCs based on broth recovery methods and minimum biofilm eradication concentrations (MBECs) of antistaphylococcal antibiotics for multilayer biofilms formed by 'biofilm-positive' S. epidermidis strains and monolayer biofilms formed by their 'biofilm-negative' mutants/variants. Bacterial densities and the quantity of persister cells in both multilayer and monolayer biofilms were assessed to evaluate their roles in biofilm resistance. Monolayer and multilayer biofilms presented similar susceptibilities to multiple antibiotics, based on biofilm MIC, broth recovery-based biofilm MBC and MBEC results. Multilayer biofilms demonstrated higher viable count-based MBCs than monolayer biofilms. Both monolayer and multilayer biofilms had very high bacterial densities of approximately 10(11-12) cfu/mL. Persister cells were found in both monolayer and multilayer biofilms, but not in planktonic cultures at log phase. The presence of persister cells in monolayer and multilayer biofilms appeared to be strain and antibiotic dependent. The adherent growth mode, rather than the ability to build up a typical multilayer biofilm structure, contributes to the high resistance of biofilms to antibiotics, and therefore might be the main virulence factor of coagulase-negative staphylococci (CoNS) with respect to antibiotic resistance. The presence of persister cells in CoNS biofilms plays an important role in antibiotic resistance. Growth at high bacterial densities is another significant factor in biofilm resistance.

Construction of In-Frame aroA Deletion Mutants of Mannheimia haemolytica , Pasteurella multocida , and Haemophilus somnus by Using a New Temperature-Sensitive Plasmid

A temperature-sensitive (TS) plasmid was generated from the endogenous streptomycin resistance plasmid of Mannheimia hemolytica and used to engineer in-frame aroA deletion mutants of Mannheimia hemolytica, Pasteurella multocida, and Haemophilus somnus. TS replacement plasmids carrying in-frame aroA deletions were constructed for each target species and introduced into host cells by electroporation. After recovery in broth, cells were spread onto plates containing antibiotic and incubated at 30 degrees C, the permissive temperature for autonomous plasmid replication. Transfer of transformants to selective plates cultured at a nonpermissive temperature for plasmid replication selected for single-crossover mutants consisting of replacement plasmids that had integrated into host chromosomes by homologous recombination. Transfer of the single-crossover mutants back to a permissive temperature without antibiotic selection drove plasmid resolution, and, depending on where plasmid excision occurred, either deletion mutants or wild-type cells were generated. The system used here represents a broadly applicable means for generating unmarked mutants of Pasteurellaceae species.